CN114752522B - Salt-tolerant bacillus for high-yield gamma-polyglutamic acid and fermentation condition optimization thereof - Google Patents
Salt-tolerant bacillus for high-yield gamma-polyglutamic acid and fermentation condition optimization thereof Download PDFInfo
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Abstract
The invention relates to a salt-tolerant bacillus for high-yield gamma-polyglutamic acid and fermentation condition optimization thereof. The invention separates a novel salt-tolerant bacillus F29 for efficiently producing gamma-polyglutamic acid from organically cultured soybeans. By optimizing the fermentation conditions, the yield of the gamma-polyglutamic acid can reach 50.9g/L, the fermentation rate is 1.36g/L/h, and the substrate conversion rate is 2.23g Gamma-polyglutamic acid /g Glutamic acid More than twice that of the existing researches. The invention utilizes salt-tolerant bacillus to produce polyglutamic acid, has strong advantages in terms of yield, productivity and conversion rate, and can greatly save production cost in industrial application.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a salt-tolerant bacillus for high-yield gamma-polyglutamic acid and optimization of fermentation conditions thereof.
Background
Gamma-polyglutamic acid (gamma-PGA), also known as natto gum or polyglutamic acid, is a polymer composed of L-glutamic acid and D-glutamic acid linked by an amide bond between an alpha-amino group (alpha-amino) and a gamma-carboxyl group (gamma-carboxyl). The molecular weight of the gamma-PGA is up to 10 megadaltons, and the gamma-PGA has excellent water solubility, super-strong adsorptivity, biodegradability and safety. The gamma-PGA is widely used as a water-retaining agent, a flocculating agent, an adsorbent, a drug carrier and the like in various aspects such as cosmetics, medicines, foods, sewage treatment, agriculture and the like.
Gamma-PGA was first found in natto, and later studies have found that various bacillus such as Bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus licheniformis (Bacillus licheniformis) and the like can synthesize gamma-PGA. At present, gamma-PGA production strains are divided into two types, one type is glutamic acid independent strains, and the strains have low gamma-PGA yield and limit the working processApplication in industrial production; the other is a glutamic acid-dependent strain, and although high yield of γ -PGA is obtained in industrial production, production cost is increased due to the addition of a large amount of glutamic acid from an external source. The yield of gamma-PGA of genetically engineered bacterium B.subtilis S18-3-vgb+ reaches 60.5g/L in the fermentation process, which is 2.07 times higher than that of wild bacterium, the fermentation rate is 0.84g/L/h, and the substrate conversion rate is about 1.5g Gamma-polyglutamic acid /g Glutamic acid (Su et al, improved poly-gamma-glutamic acid production by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis technology 2010;101 (12): 4733-4736), which is the highest conversion reported in the literature. The leading biological agriculture Co., ltd, utilizes bacillus licheniformis to produce polyglutamic acid with the yield of 38-44g/L and the production rate of 1.03-1.47g/L/h, but the conversion rate is lower than 1.0g Gamma-polyglutamic acid /g Glutamic acid (CN 104694437 B.2018.04.27)。
Although the production of the gamma-PGA has been greatly progressed, the problems of long fermentation period, low production rate, low conversion rate and high production cost exist, and the variety of the gamma-PGA production strains used in the industry is relatively small at present. Therefore, the new gamma-PGA production strain is screened, the fermentation condition is optimized, the yield and the conversion rate are improved, the production cost is reduced, and the method has great industrial application value.
Disclosure of Invention
The invention aims to solve the technical problems of providing a high-conversion rate strain for efficiently producing gamma-PGA and a method for producing gamma-PGA by fermentation, which have the advantages of short fermentation period, high substrate conversion rate, high product yield and low production cost, and lay a foundation for industrial application.
An object of the present invention is to provide a salt-tolerant bacillus strain.
The preservation number of the salt-tolerant bacillus (Bacillus halotolerans) F29 provided by the invention is CGMCC NO.23662.
The use of the aforementioned salt-tolerant bacillus or bacterial suspension thereof or culture solution thereof or fermentation solution thereof in at least one of the following 1) to 3) or in a product having at least one of the following 1) to 3) is also within the scope of protection of the present invention:
1) Producing gamma-polyglutamic acid;
2) Improving the yield of gamma-polyglutamic acid;
3) The conversion rate of gamma-polyglutamic acid is improved.
It is another object of the present invention to provide a method for producing gamma-polyglutamic acid.
The method provided by the invention comprises the following steps: fermenting the salt-tolerant bacillus to obtain the gamma-polyglutamic acid.
The fermentation adopts a culture medium containing glutamic acid;
in an embodiment of the invention, glutamic acid is present in the form of sodium glutamate.
The fermentation adopts a culture medium, wherein a C source in the culture medium is sucrose, glucose, fructose, glycerol, xylose or arabinose;
and/or the N source in the fermentation adoption culture medium is ammonium chloride, ammonium sulfate, peptone, tryptone or yeast powder.
In the above method, the fermentation temperature is 28-42 ℃.
In the above method, the pH value of the fermentation is 5.0-8.5.
In the above method, the fermentation process further comprises the following steps: sucrose is added to the fermentation system when the total sugar concentration in the fermentation system is lower than 10g/L.
The fermentation medium comprises sucrose, sodium glutamate, ammonium sulfate, and K 2 HPO 4 、MgSO 4 ·7H 2 O、 FeCl 3 ·6H 2 O、CaCl 2 、MnSO 4 ·H 2 O and NaCl.
In the initial fermentation medium, the sucrose (3%), sodium glutamate (2%), ammonium sulfate (0.5%), K 2 HPO 4 (0.05％)、MgSO 4 ·7H 2 O(0.05％)、FeCl 3 ·6H 2 O(0.0004％)、CaCl 2 (0.015％)、MnSO 4 ·H 2 O (0.0104%) and NaCl (0.05%),% are mass percent.
The optimal fermentation medium per L is: 77.58g of sucrose, 43.02g of sodium glutamate and K 2 HPO 4 2.23g, the rest is 5g of ammonium sulfate and MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 ·H 2 0.104g of O, 0.5g of NaCl and distilled water are dissolved and the volume is fixed to 1L.
Optimal fermentation conditions: the initial pH of the fermentation medium is 5.5, and the culture temperature is 37 ℃.
The invention also provides a method for improving the conversion rate of gamma-polyglutamic acid, which comprises the steps of the method.
The invention also provides a product comprising the salt-tolerant bacillus or the bacterial suspension thereof or the culture solution thereof or the fermentation solution thereof.
The product has at least one of the following functions:
1) Producing gamma-polyglutamic acid;
2) Improving the yield of gamma-polyglutamic acid;
3) The conversion rate of gamma-polyglutamic acid is improved.
The invention separates a novel salt-tolerant bacillus F29 for efficiently producing gamma-polyglutamic acid from organically cultured soybeans. By optimizing the fermentation conditions, the yield of the gamma-polyglutamic acid can reach 50.9g/L, the fermentation rate is 1.36g/L/h, and the substrate conversion rate is 2.23g Gamma-polyglutamic acid /g Sodium glutamate More than twice that of the existing researches. The invention utilizes salt-tolerant bacillus to produce polyglutamic acid, has strong advantages in terms of yield, productivity and conversion rate, and can greatly save production cost in industrial application.
Preservation description
Strain name: salt-tolerant bacillus
Latin name: bacillus halotolerans
Strain number: f29 (F29)
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation date: 2021, 10, 25
Accession numbers of the preservation center: CGMCC No.23662
Drawings
FIG. 1 is a diagram showing the morphology of cells and colonies: a is a single cell form under a 100 times microscope; b is colony morphology on the plate medium.
FIG. 2 shows the evolution tree of salt tolerant bacillus F29.
FIG. 3 is a graph showing the effect of carbon source on the production of gamma-polyglutamic acid by a strain.
FIG. 4 shows the effect of nitrogen source on the production of gamma-polyglutamic acid by a strain.
FIG. 5 shows the effect of pH on the production of gamma-polyglutamic acid by a strain.
FIG. 6 is a graph showing the effect of temperature on the production of gamma-polyglutamic acid by a strain.
Fig. 7 is a 3D graph of sucrose and sodium glutamate interaction response.
FIG. 8 is a 3D plot of the interaction response of sucrose and dipotassium hydrogen phosphate.
FIG. 9 is a 3D graph of the interaction response of sodium glutamate and dipotassium hydrogen phosphate.
FIG. 10 is a feed fermentation curve of B.halodurans F29 in a 5L fermenter.
FIG. 11 shows a gamma-polyglutamic acid standard curve.
FIG. 12 is a standard curve of L-glutamic acid.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following describes the objects, technical schemes and advantageous effects of the present invention in detail with reference to examples, so as to facilitate understanding of the skilled person.
Example 1 isolation screening and identification of gamma-polyglutamic acid-producing Bacillus halodurans F29
1. Separation and screening of gamma-polyglutamic acid-producing bacillus halodurans F29
1. Organically grown soybeans were purchased from the Dong Wudao vegetable market in the free-rise economic area of Dong Li district, tianjin, city. Firstly, 10g of soybean is weighed and added into 90mL of sterile water, and the soybean is boiled in a boiling water bath for 5 minutes to remove part of non-spore strain, and the soybean is cooled at room temperature for standby.
2. Taking 1mL of cooled liquid, and placing into a test tube containing 9mL of sterile water to obtain 10 -1 Diluting the bacterial liquid from 10 -1 1mL of the diluted bacterial liquid is taken and put into a test tube containing 9mL of sterile water, namely 10 -2 The dilution of the bacterial liquid is similarly 10 -3 、10 -4 Bacterial liquid of dilution.
3. Preparing a separation medium: 5g of yeast powder, 10g of tryptone, 10g of sodium chloride, 20g of glucose, 10g of sodium glutamate, 20g of agar and distilled water are dissolved and the volume is fixed to 1L; sterilizing at 115 deg.c for 20min at pH 6.8-7.2.
The plates were poured, approximately 20mL of medium was poured into each dish, the plates were allowed to stand, and the plates were allowed to cool and solidify for use.
4. Diluting the bacterial liquid 10 -1 、10 -2 、10 -3 、10 -4 Each of the plates was plated with 5 dishes per gradient.
5. The culture dish is inverted and placed in a 37 ℃ incubator for culturing for 24 to 48 hours.
6. Selecting single colony with surface fold and viscosity by using a sterile inoculating needle, and streaking and culturing on a new solid plate; the single colony after purification can be obtained after 3 times of purification.
7. The purified strain obtained in the step 6 was inoculated into a glass test tube containing 3mL of liquid seed medium, cultured at 37℃at 200rpm for 12 hours, and then inoculated into a 250mL shake flask containing 50mL of fermentation medium in an inoculum size of 1%, and fermented at 37℃at 200rpm/min for 24 hours.
The yield of gamma-polyglutamic acid was determined using CTAB color development. Strain F29 produced the highest concentration of gamma-polyglutamic acid.
The formula of the liquid seed culture medium comprises the following components: 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, 20g of glucose and 10g of sodium glutamate, and dissolving the mixture in single distilled water and fixing the volume to 1L; the pH is 6.8-7.2; sterilizing at 115 deg.C for 20min.
The formula of the fermentation medium comprises the following components: glucose30g, ammonium chloride 5g, sodium glutamate 20g, K 2 HPO 4 0.5g， MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 ·H 2 0.104g of O, 0.5g of NaCl, and dissolving with single distilled water and fixing the volume to 1L; the pH is 6.8-7.2; sterilizing at 115 deg.C for 20min.
2. Identification of gamma-polyglutamic acid-producing bacillus halodurans F29
1. Morphological identification
The cells are rod-shaped, and the cell size is 0.3-1 μm. The colony is white, the colony is larger, the surface is convex, smooth and thick, the edge is wrinkled, gram positive and spore is produced. The morphology of the cells and colonies is shown in FIG. 1.
2. Molecular characterization
The strain F29 which can efficiently synthesize gamma-polyglutamic acid and is obtained by screening, extracting genome and amplifying by using 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (TACGGCTACCTTGTTACGACTT) primers to obtain a 16S rDNA sequence.
The 16S rDNA sequence was analyzed and the results are shown in SEQ ID No. 1.
The above sequence 1 was aligned with the model strain on EzBioCloud and a treeing was constructed using the Neighbor-Joining algorithm using MEGA7 software (FIG. 2). F29 and Bacillus halotolerans ATCC25096 are in the same branch, and F29 is identified as salt-tolerant bacillus Bacillus halotolerans by combining the previous morphological identification results.
The strain F29 is preserved in China general microbiological culture collection center (CGMCC) for 10 and 25 days in 2021, with the preservation number of CGMCC NO.23662 and the classification of Bacillus halotolerans.
Example 2 optimization of fermentation conditions for salt tolerant bacillus F29
1. Single factor condition fumbling experiment for producing gamma-polyglutamic acid by fermenting salt-tolerant bacillus F29
The single colony of the salt-tolerant bacillus F29 obtained in the example 1 is inoculated into a 250mL shaking flask filled with 50mL of seed culture medium, and is cultured for 12 hours at the temperature of 37 ℃ and the rotating speed of 200rpm/min, so that the salt-tolerant bacillus F29 seed solution is obtained.
1. Influence of different carbon sources on production of gamma-polyglutamic acid by salt-tolerant bacillus F29
The F29 seed solution was inoculated at 1% inoculum size into 250mL shake flasks containing 50mL of different C-source fermentation media and fermented at 37℃at 200rpm/min for 24h.
The fermentation medium formula of the different C sources comprises: 30g of C source, 5g of ammonium chloride, 20g of sodium glutamate and K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 ·H 2 0.104g of O, 0.5g of NaCl, and dissolving with single distilled water and fixing the volume to 1L; pH 7.2; sterilizing at 115 deg.C for 20min.
The C source is sucrose, glucose, fructose, glycerol, xylose or arabinose.
The yield of gamma-polyglutamic acid was determined by using a CTAB color development method as follows:
(1) Preparing gamma-polyglutamic acid standard solution: 0.1g of gamma-polyglutamic acid standard (Shanghai Michelin Biochemical technology Co., ltd.; CAS number: 25513-46-6) was weighed out accurately, dissolved in distilled water in a 100mL volumetric flask and fixed in volume, to obtain a standard solution of 1 g/L. And carrying out gradient dilution to obtain gamma-polyglutamic acid standard solution with the concentration of 0.1g/L,0.2g/L,0.3g/L,0.4g/L and 0.5g/L in sequence.
(2) Purification of gamma-polyglutamic acid: centrifuging the fermented liquid obtained by fermentation at 12000r/min for 10min, removing thalli, taking 100 mu L of supernatant, adding into 4% (w/v, g: mL) trichloroacetic acid (TCA) solution containing 900 mu L, heating at 50 ℃ for 10min to remove protein in the solution, centrifuging at 12000r/min for 10min, and taking the supernatant to obtain the prepared sample liquid. The assay was performed after dilution by appropriate factors.
(3) Preparation of CTAB solution: a2% (w/v, g: mL) NaOH solution was prepared, and CTAB was added to a final concentration of 0.07M using this as a solvent.
(4) CTAB turbidimetry assay: accurately taking 50 mu L of standard solution or sample solution, adding into a 1.5mL centrifuge tube filled with 450 mu L of distilled water, accurately adding 500 mu L of CTAB test solution, fully mixing, and standing for 3min. The reaction solution was poured into a 1mm cuvette, and absorbance at a wavelength of 400nm (A400) was measured, and 500. Mu.L of distilled water was added as a blank. The standard curve obtained is shown in fig. 11.
The concentration of the gamma-polyglutamic acid in the fermentation liquor is OD400/0.9615 multiplied by dilution
The method for detecting biomass is as follows: the seed solutions were inoculated into 3 flasks containing 30ml of fermentation medium, cultured for 24 hours, and then absorbance at 600nm (OD 600) was measured, centrifuged at 5000rpm for 20 minutes, and the cells were collected, washed three times with distilled water, freeze-dried and weighed. The average OD600 of the cells obtained was 12.47, the average dry weight of the cells was 0.155g, and 1 od=0.414 g dry weight of the cells was calculated. The biomass calculating method comprises the following steps: dilution of the broth to the appropriate multiple, absorbance (OD 600) at 600nm wavelength, biomass = OD600 x 0.414 x dilution
The results of the gamma-polyglutamic acid and biomass detection are shown in FIG. 3, the best carbon source for producing gamma-polyglutamic acid by the salt-tolerant bacillus F29 is sucrose, and the yield of the gamma-polyglutamic acid is 8.05g/L.
2. Influence of different nitrogen sources on production of gamma-polyglutamic acid by salt-tolerant bacillus F29
The F29 seed solution was inoculated at 1% inoculum size into 250mL shake flasks containing 50mL of different N sources of fermentation medium and fermented at 37℃at 200rpm/min for 24h.
The formula of the fermentation medium with different N sources comprises the following components: 30g of sucrose, 5g of N source, 20g of sodium glutamate and K 2 HPO 4 0.5g， MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 ·H 2 0.104g of O, 0.5g of NaCl, and dissolving with single distilled water and fixing the volume to 1L; pH 7.2; sterilizing at 115 deg.C for 20min.
The N source is ammonium chloride, ammonium sulfate, peptone, tryptone or yeast powder.
The yield of gamma-polyglutamic acid was determined using CTAB color development.
Biomass is detected.
The results of the gamma-polyglutamic acid and biomass detection are shown in FIG. 4, and the best nitrogen source for producing gamma-polyglutamic acid by the salt tolerant bacillus F29 is ammonium sulfate, and the yield of the gamma-polyglutamic acid is 9.65g/L.
3. Effect of different pH on production of gamma-polyglutamic acid by salt-tolerant Bacillus F29
The F29 seed solution was inoculated into 250mL shake flasks containing 50mL of fermentation media having different pH values at an inoculum size of 1%, and fermented at 37℃at 200rpm/min for 24 hours.
The formula of the fermentation medium with different pH values comprises the following components: 30g of sucrose, 5g of ammonium sulfate, 20g of sodium glutamate and K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 ·H 2 0.104g of O, 0.5g of NaCl, and dissolving with single distilled water and fixing the volume to 1L; the pH of the fermentation medium is adjusted to be pH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0 and pH8.5 respectively, so as to obtain the fermentation medium with different pH values. Sterilizing at 115 deg.C for 20min.
The yield of gamma-polyglutamic acid was determined using CTAB color development.
Biomass is detected.
As shown in FIG. 5, the results of the detection of gamma-polyglutamic acid and biomass revealed that the optimum pH for producing gamma-polyglutamic acid by Bacillus halodurans F29 was 5.5 and the yield of gamma-polyglutamic acid was 11.26g/L.
4. Influence of different temperatures on production of gamma-polyglutamic acid by salt-tolerant bacillus F29
The fermentation temperatures for the production of gamma-polyglutamic acid by the strain were set to 4 values of 28℃at 32℃at 37℃and 42℃respectively.
The F29 seed solutions were inoculated in an inoculum size of 1% into 250mL shake flasks containing 50mL of fermentation medium, and fermented at 28℃at 32℃at 37℃and 42℃at 200rpm/min, respectively, for 24 hours.
The formula of the fermentation medium comprises the following components: 30g of sucrose, 5g of ammonium sulfate, 20g of sodium glutamate and K 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 ·H 2 0.104g of O, 0.5g of NaCl, and dissolving with single distilled water and fixing the volume to 1L; the pH of the fermentation medium was adjusted to 5.5. Sterilizing at 115 deg.C for 20min.
The yield of gamma-polyglutamic acid was determined using CTAB color development.
Biomass is detected.
As shown in FIG. 6, the optimal temperature for producing gamma-polyglutamic acid by Bacillus halodurans F29 was 37℃and the yield of gamma-polyglutamic acid was 9.10g/L.
2. Optimization experiment of salt-tolerant bacillus F29 for producing gamma-polyglutamic acid
Plackett-Burman experimental design screening for significant influencing factors
Sucrose, ammonium sulfate, sodium glutamate, K 2 HPO 4 ，MgSO 4 ·7H 2 O，FeCl 3 ·6H 2 O，CaCl 2 ， MnSO 4 ·H 2 O and NaCl 9 influencing factors, two levels of each factor were combined for experiments using Design Expert 10.0.4 software, for a total of 12 experiments, as shown in table 1. The concentrations of each factor at two levels and the analysis of variance results are shown in table 2. Three experiments are carried out on each experimental combination, the inoculation amount is 1%, the culture temperature is 37 ℃, the initial pH is 5.5, the rotation speed of a shaking table is 200rpm/min, the culture is carried out for 24-48 hours, the concentration of gamma-polyglutamic acid is detected in different time periods, and the highest concentration of each combination is taken as a response value.
Table 1 shows the combinations and results of Plackett-Burman experiments
Table 2 shows the true concentrations of the influencing factors at two levels and the analysis of variance results
From the analysis of variance results of Table 2, the model reached a significant level (P<0.05)。Sucrose, sodium glutamate, ammonium sulfate, K 2 HPO 4 、FeCl 3 ·6H 2 O and CaCl 2 ·2H 2 O has a significant effect on the production of gamma-polyglutamic acid (P<0.05). Sucrose, sodium glutamate, K 2 HPO 4 And FeCl 3 ·6H 2 O has a positive correlation with the production of gamma-polyglutamic acid, indicating that the higher the concentration, the higher the production of gamma-polyglutamic acid. Ammonium sulfate and CaCl 2 ·2H 2 O has a negative correlation with polyglutamic acid production, indicating that the higher the concentration, the lower the gamma-polyglutamic acid production. FeCl 3 ·6H 2 The P value of O was 0.0443, close to 0.05, indicating little effect. Thus, sucrose, sodium glutamate and K are selected 2 HPO 4 And carrying out response surface analysis design experiments.
Design of face-to-face center composite point experiment
Sucrose, sodium glutamate and K were selected according to Plackett-Burman experimental results 2 HPO 4 As independent variables, the yield of gamma-polyglutamic acid is taken as a response value, a Design-Expert 10.0.4 software is applied to Design a three-factor three-level response surface analysis experiment by using a Face-to-Face center complex point experimental principle, the specific Design and the corresponding yield of gamma-polyglutamic acid are shown in Table 3 in detail, and the composition of the optimized culture medium is determined according to the analysis result of the investigation factors and a response surface three-dimensional model.
Table 3 shows the experimental factor level and coding level table of the Face-to-Face center complex point and the experimental result
The analysis of variance of the design experiment results of the Face-to-Face center composite points is shown in Table 4.
Table 4 shows the analysis of variance of the design experiment results of the Face-to-Face center composite points
According to the experimental results in table 4, the software performs analysis and fitting to obtain a polynomial regression equation, and the obtained fitting regression equation of the gamma-polyglutamic acid yield is:
Y＝20.05-0.48X 1 +0.65X 2 +0.37X 4 +0.72X 1 X 4 -2.59X 1 2 -2.25X 2 2 -0.74X 4 2
the analysis of variance of the fit equation is shown in Table 4, and from Table 4, the model reached a significant level (P<0.05). P= 0.4376 of the mismatch term>And 0.05, the difference of the mismatch terms is not obvious, so that the error in the experimental process is small, and the regression equation has good fitting degree to the experiment. Regression coefficient R of model 2 = 0.9834, demonstrating that the model is reliable in analysis and prediction of gamma-polyglutamic acid production.
3. Response surface optimization of culture medium composition
The response surface 3D graphics facilitate direct observation of the relationship between individual variable values and response values and interactions between the two variables. From FIGS. 7-9, it can be seen that the effect of sucrose, sodium glutamate and dipotassium hydrogen phosphate interactions on gamma-polyglutamic acid production. The response value Y reaches the highest value along with the increase of each factor, and then gradually decreases, which indicates that each factor has an optimal value for the yield of gamma-polyglutamic acid, and the inhibition effect is generated when the response value Y exceeds the optimal value, and the response value Y is identical with the negative value of the square term coefficient of each factor in regression analysis results. For each influencing factor, the steeper the curve, the more sensitive the response face value Y is to changes in the factor, and the greater the influence of this factor interaction on the production of gamma-polyglutamic acid; whereas the more gradual the slope of the surface, the less the change in factor affects the response. The effect of sucrose and sodium glutamate on the production of gamma-polyglutamic acid was greatest, and the regression analysis results of Table 4 were also consistent with the P values of sucrose and sodium glutamate being 0.0089 and 0.0014, respectively, which were much less than 0.05.
From the regression equation, each 1L of optimized fermentation medium for gamma-polyglutamic acid was obtained as follows: 77.58g of sucrose, glutamineSodium acid 43.02g, K 2 HPO 4 2.23g, the rest is 5g of ammonium sulfate and MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 ·H 2 O0.104 g, naCl 0.5g, distilled water to 1L, initial pH5.5, culture temperature 37 ℃. The maximum yield of gamma-polyglutamic acid is 20.16g/L.
4. Optimized media validation
The salt-tolerant bacillus F29 seed solution obtained in the previous step was inoculated into a 250mL shaking flask containing 50mL of the optimized fermentation medium obtained in the previous step 3, and the inoculated amount was 1%, and the fermentation was performed at 37℃and 200rpm/min for 48 hours, and three repeated experiments were performed.
The yield of gamma-polyglutamic acid was determined by CTAB color development, and as a result, the yield of gamma-polyglutamic acid was 20.67. 20.67 g/L. The actual value is very close to the theoretical value, which shows that the regression model can well reflect the influence of the concentration of each component of the culture medium on the yield of the gamma-polyglutamic acid, and proves the feasibility of improving the yield of the gamma-polyglutamic acid by adopting a culture medium optimization method.
Example 3 further enhancement of gamma-polyglutamic acid production and yield
1. Solid plate culture
The salt-tolerant bacillus F29 obtained in the example 1 is inoculated on a solid plate and cultured for 12 hours at 37 ℃ to obtain an activated solid culture strain for later use.
The formula of the solid plate culture medium is as follows: 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, 20g of glucose, 10g of sodium glutamate and 20g of agar powder, and dissolving the mixture in single distilled water and fixing the volume to 1L; the pH is 6.8-7.2.
2. Shake flask seed culture
And (2) taking the solid culture strain prepared in the step (1), selecting a single colony, inoculating the single colony into a 250mL shake flask filled with 50mL of liquid culture medium, and culturing for 12 hours at 37 ℃ at 200rpm/min to obtain shake flask seed liquid for later use.
The liquid culture medium formula is consistent with the solid plate culture medium formula, and agar powder is not added.
3. Fermentation tank culture
Inoculating the seed solution cultured in the step 2 to 5L of fermentation tank containing the optimized fermentation medium obtained in the example 2, fermenting, wherein the liquid loading amount of the optimized fermentation medium is 3L, the inoculation amount is 5% of the fermentation volume, the culture temperature is 37 ℃, the stirring speed is 600rpm/min (BIOTECH-5 JG; shanghai Baozhen biological equipment engineering Co., ltd.) and the aeration rate is maintained at 2vvm for 12h and at 3vvm after 0-12h, so that the dissolved oxygen is maintained at more than 5% by controlling the rotating speed and the aeration rate. When the sucrose concentration is lower than 10g/L, one bottle of sucrose solution is added at a time. After culturing for 48 hours, collecting fermentation liquor, and obtaining fermentation liquor containing gamma-polyglutamic acid.
The fermentation medium described above is the optimized medium described in example 3.
The sucrose solution composition of the feed is as follows: 100g sucrose was added with 50mL water and the mixture was placed in 250mL shake flasks and sterilized at 115℃for 20min.
And (3) measuring the yield of gamma-polyglutamic acid in fermentation liquor at different fermentation times by adopting a CTAB chromogenic method.
Biomass is detected.
L-glutamic acid was detected by the following method:
standard substance treatment: 0.1g of L-glutamic acid standard (Beijing Soy Bao technology Co., ltd.; CAS: 56-86-0) was weighed accurately, dissolved in distilled water in a 100mL volumetric flask, and the volume was fixed, to obtain 1g/L of L-glutamic acid standard solution. And carrying out gradient dilution to obtain 0.2g/L,0.4g/L,0.6g/L and 0.8 g/L-glutamic acid standard solution in sequence. The standard solution was passed through a 0.2 μm filter membrane for use.
Sample treatment:
amino acid detection method: the fermentation broth obtained by fermentation was centrifuged at 12000r/min for 10min, 50. Mu.L of the supernatant was taken, and added to distilled water containing 950. Mu.L, followed by mixing. Pass through a 0.2 μm filter membrane for use.
An Agilent 1260 affinity liquid chromatograph equipped with a quaternary pump, liquid autosampler, column oven and DAD detector. The column used was Agilent ZORBAX Eclipse Plus C (4.6X105 mm,3.5 μm).
Derivatizing agents OPA and FMOC were purchased from agilent company.
Mobile phase a) 10mmol/L disodium hydrogen phosphate and 10mM sodium borate solution, pH adjusted to 8.2 with hydrochloric acid; b) Methanol to acetonitrile to water, 45:45:10 (volume ratio v: v).
The automatic sample injection program is as follows: 1) Aspirate 2.5 μl borate buffer; 2) Aspirate 1.0 μl sample; 3) Mixing 3.5 mu L of the mixed solution for 5 times at a cleaning port; 4) Wait for 0.2 minutes; 5) Aspirate 0.5 μl OPA; 6) Mixing 4 mu L of the mixed solution for 10 times at a cleaning port; 7) Aspirate 0.4 μl FMOC; 8) Mixing 4.4 mu L of the mixed solution for 10 times at a cleaning port; 9) Aspirate 32 μl of diluent; 10 20. Mu.L of the mixture was mixed 8 times at the washing port; 11 Sample introduction; 12) Wait for 0.1 min; 13 A) the valve switches to the bypass.
The gradient procedure is:
a detector: 338nm. The column temperature was 40 ℃.
From the standard concentration and peak area, a standard curve (fig. 12) was obtained as follows:
the content of L-glutamic acid in the sample is: peak area/1476 x dilution.
An Agilent 1260 affinity liquid chromatograph equipped with a quaternary pump, liquid autosampler, column oven and RID detector. The chromatographic column selected was a Berle Aminex HPX-87P chromatographic column (300X 78mm;9 μm). The mobile phase is chromatographic grade pure water; the column temperature was 65 ℃. Sampling time is 20min, and sampling volume is 20 mu L. And (3) manufacturing a standard curve by using sucrose, fructose and glucose standard substances, and calculating the content of each sugar in the fermentation broth according to the peak area and the standard curve, thereby obtaining the total sugar content.
The maximum fermentation rate is: the yield of gamma-PGA reached 48.98g/L at 36h, and the fermentation rate was 48.98/36=1.36 g/L/h.
The conversion rate calculation method comprises the following steps: at 48h, initial glutamic acid 43.68g/L, 20.93g/L remained after fermentation, and consumption of 22.75g/L, 50.91g/L of gamma-PGA was produced. The conversion of glutamic acid to γ -PGA was 50.91/22.75=2.23 g/g.
As a result, as shown in FIG. 10, it was revealed that the production of gamma-polyglutamic acid could be 50.9g/L at 48 hoursThe maximum fermentation rate is 1.36g/L/h, and the conversion rate is 2.23g Gamma-polyglutamic acid /g Glutamic acid 。
Finally, it is noted that the above preferred embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail by means of the above preferred embodiments, it will be apparent to those skilled in the art that various changes in form and details may be made therein without departing from the scope of the present invention.
SEQUENCE LISTING
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> salt-tolerant bacillus for high-yield gamma-polyglutamic acid and fermentation condition optimization thereof
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1425
<212> DNA
<213> Bacillus halotolerans
<400> 1
ggcggctggc tccataaagg ttacctcacc gacttcgggt gttacaaact ctcgtggtgt 60
gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta 120
ctagcgattc cagcttcacg cagtcgagtt gcagactgcg atccgaactg agaacagatt 180
tgtgggattg gcttaacctc gcggtttcgc tgccctttgt tctgtccatt gtagcacgtg 240
tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc ctccggtttg 300
tcaccggcag tcaccttaga gtgcccaact gaatgctggc aactaagatc aagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc atgcaccacc 420
tgtcactctg cccccgaagg ggacgtccta tctctaggat tgtcagagga tgtcaagacc 480
tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 540
ccgtcaattc ctttgagttt cagtcttgcg accgtactcc ccaggcggag tgcttaatgc 600
gttagctgca gcactaaggg gcggaaaccc cctaacactt agcactcatc gtttacggcg 660
tggactacca gggtatctaa tcctgttcgc tccccacgct ttcgctcctc agcgtcagtt 720
acagaccaga gagtcgcctt cgccactggt gttcctccac atctctacgc atttcaccgc 780
tacacgtgga attccactct cctcttctgc actcaagttc cccagtttcc aatgaccctc 840
cccggttgag ccgggggctt tcacatcaga cttaaggaac cgcctgcgag ccctttacgc 900
ccaataattc cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta 960
gccgtggctt tctggttagg taccgtcaag gtaccgccct attcgaacgg tacttgttct 1020
tccctaacaa cagagcttta cgatccgaaa accttcatca ctcacgcggc gttgctccgt 1080
cagactttcg tccattgcgg aagattccct actgctgcct cccgtaggag tctgggccgt 1140
gtctcagtcc cagtgtggcc gatcaccctc tcaggtcggc tacgcatcgt tgccttggtg 1200
agccattacc tcaccaacta gctaatgcgc cgcgggtcca tctgtaagtg gtagccgaag 1260
ccacctttta tgtttgaacc atgcggttca aacaagcatc cggtattagc cccggtttcc 1320
cggagttatc ccagtcttac aggcaggtta cccacgtgtt actcacccgt ccgccgctaa 1380
catcagggag caagctccca tctgtccgct cgactgcatg tatag 1425
Claims (8)
1. Salt-tolerant bacillus(Bacillus halotolerans）F29 with the preservation number of CGMCC NO.23662.
2. Use of a salt tolerant bacillus or a bacterial suspension thereof or a culture broth thereof or a fermentation broth thereof in at least one of the following 1) -3) or in a product having at least one of the following 1) -3):
1) Producing gamma-polyglutamic acid;
2) Improving the yield of gamma-polyglutamic acid;
3) The conversion rate of gamma-polyglutamic acid is improved;
the salt-tolerant bacillus is the salt-tolerant bacillus of claim 1.
3. A method for producing gamma-polyglutamic acid, characterized by: fermenting the salt-tolerant bacillus of claim 1 to obtain gamma-polyglutamic acid.
4. A method according to claim 3, characterized in that:
the fermentation adopts a culture medium containing glutamic acid;
the fermentation adopts a culture medium, wherein a C source in the culture medium is sucrose, glucose, fructose, glycerol, xylose or arabinose;
and/or the N source in the fermentation adoption culture medium is ammonium chloride, ammonium sulfate, peptone, tryptone or yeast powder.
5. A method according to claim 3 or 4, characterized in that: the fermentation temperature is 28-42 ℃.
6. A method according to claim 3 or 4, characterized in that: the pH value of the fermentation is 5.0-8.5.
7. A method according to claim 3 or 4, characterized in that:
the fermentation process further comprises the following steps: sucrose is added to the fermentation system when the total sugar concentration in the fermentation system is lower than 10g/L.
8. A method for increasing the conversion rate of gamma-polyglutamic acid, comprising the steps of:
1) Plate culture of solid
Inoculating the salt-tolerant bacillus of claim 1 on a solid plate culture medium, and culturing for 12 hours at 37 ℃ to obtain an activated solid culture strain;
2) Seed culture in shake flask
Inoculating the solid culture strain into a liquid culture medium, and culturing at 37 ℃ and 200rpm/min for 12 hours to obtain shake flask seed liquid;
3) Culturing in a fermentation tank
Inoculating the seed liquid into a fermentation tank containing a fermentation medium for fermentation, wherein the liquid loading amount of the fermentation medium is 3L, the inoculation amount is 5% of the fermentation volume, the culture temperature is 37 ℃, the stirring speed is 600rpm/min, the aeration rate is maintained at 2vvm for 0-12h, and the aeration rate is maintained at 3vvm after 12h, so that the dissolved oxygen is maintained at more than 5% by controlling the rotating speed and the aeration rate; when the concentration of the sucrose is lower than 10g/L, one bottle of sucrose solution is added at a time; after culturing for 48 hours, collecting fermentation liquor, namely obtaining fermentation liquor containing gamma-polyglutamic acid;
the formula of the solid plate culture medium is as follows: 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, 20g of glucose, 10g of sodium glutamate and 20g of agar powder, and dissolving the mixture in single distilled water and fixing the volume to 1L; the pH is 6.8-7.2;
the liquid culture medium formula is consistent with the solid plate culture medium formula, and agar powder is not added;
the fermentation medium comprises the following formula per 1L: 77.58g of sucrose, 43.02g of sodium glutamate and K 2 HPO 4 2.23g, the rest is 5g of ammonium sulfate and MgSO 4 •7H 2 O 0.5g，FeCl 3 •6H 2 O 0.04g，CaCl 2 0.15g，MnSO 4 •H 2 0.104g of O, 0.5g of NaCl, and distilled water to 1L;
the additional sucrose solution composition was: 100g sucrose was added with 50mL water and the mixture was placed in 250mL shake flasks and sterilized at 115℃for 20min.
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